含硅二氧化钛纳米管层的体外生物活性

In vitro bioactivity of silicon-incorporated titanium dioxide nanotubes

背景:钛合金长期以来被用作可植入的生物材料,通过在其表面制备二氧化钛纳米管层可提高材料表面与骨的接触面积,然而将硅元素注入纳米管层表面是否可以进一步提高材料生物活性,有待进一步研究。目的:探讨钛表面含硅二氧化钛纳米管层对成骨细胞MC3T3-E1细胞活性的影响,为医用钛金属生物管层的改良提供实验依据。方法:利用阳极氧化法技术在钛表面制备二氧化钛纳米管层,采用等离子体浸没离子植入和沉积法,将硅离子沉积在二氧化钛纳米管层表面(实验组),以二氧化钛纳米管层为阳性对照组,光滑钛片作为阴性对照组。用扫描电镜观察各组形貌,X射线衍射仪测定各组元素组成。3组管层表面分别种植MC3T3-E1细胞,检测第1,3及5天各组细胞黏附铺展、增殖情况,第7天各组细胞相关基因的表达及第3,4周各组细胞的钙沉积情况。结果与结论:(1)扫描电镜下显示硅元素的加入没有改变管层的表面形貌,且第1天仅实验组细胞上可以观察到有丝状伪足扩展,第3天丝状伪足活性增加并开始合并,第5天丝状网络结构较其他两组明显增多;(2)X射线衍射仪显示实验组出现了对应为硅元素特征峰;(3)MTS法显示培养第1,3天,黏附于实验组及阳性对照组表面的细胞数量比阴性对照组显著增多,第5天,实验组上的细胞增殖显著高于对其他2组(P<0.05);(4)实时定量RT-PCR检测结果显示7 d时实验组中成骨相关基因Ⅰ型胶原蛋白,碱性磷酸酶,Runx2,骨钙素和骨桥蛋白基因表达均高于对照组(P<0.05);(5)第3,4周实验组和阳性对照组上的细胞与阴性对照组相比钙矿物结节形成明显升高,第4周含实验组沉积的钙矿物比其他2组显著增加(P<0.05);(6)结果提示,二氧化钛纳米管层可以通过等离子体浸没离子植入和沉积法加载硅离子并具备良好的成骨活性。

BACKGROUND:Titanium alloy has been used as an implantable biomaterial for a long time.The contact area between the material surface and bone can be improved by the preparation of TiO2 nanotube film on the surface of titanium alloys.However,further research is warranted to improve the bioactivity of silicon on the surface of the nanotube.OBJECTIVE:To investigate the effect of silicon-incorporated TiO2 nanotubes on the bioactivity of MC3T3-E1 cells in vitro and to provide experimental evidence for the biochemical modification of titanium.METHODS:Silicon-incorporated TiO2 nanotubes films on titanium surface were prepared by anodization and silicon plasma immersion ion implantation.Si-TiO2-NTs film was served as experimental group;TiO2-NTs was served as positive control group,and titanium film was used as negative control group.Field emission scanning electron microscope and X-ray photoelectron spectroscopy were used to detect the morphology and element composition of samples in three groups.MC3T3-E1 cells were seeded onto the specimen surface of these three groups.After 1,3,5 days of cultivation,cell adhesion,spreading and proliferation were detected.After 7 days of cultivation,cell-related gene expression was detected in each group.After 3 and 4 weeks of cultivation,calcium deposition of cultured cells on the samples was evaluated by alizarin red s staining.RESULTS AND CONCLUSION:(1)Under the scanning electron microscope,the addition of silicon element did not change the surface morphology of the nanotube film.On the 1st day,filopodia expansion was only observed on the cells of the experimental group;on the 3rd day,the activity of the filopodia increased and began to merge;on the 5th day,the filamentous network structure increased significantly compared with the other two groups.(2)Results from X-ray photoelectron spectroscopy showed that the characteristic peak of silicon element appeared in the experimental group.(2)At 1 and 3 days of culture,the number of adherent cells in the experimental and positive control groups was higher than that in the negative control group.At 5 days of culture,cell proliferation in the experimental group was significantly higher than that in the other two groups(P<0.05).(4)Real-time quantitative RT-PCR results showed that the expression levels of osteogenic related genes,type I collagen,alkaline phosphatase,Runx2,osteocalcin and osteopontin,were all higher in the experimental group than the control groups(P<0.05).(5)Compared with the negative control group,the cells on TiO2-NTs and Si-TiO2-NTs showed an elevated calcium mineral nodule formation at 3 and 4 weeks of culture(P<0.05).Moreover,the deposited calcium mineral on Si-TiO2-NTs exhibited a significant increase compared with the other two groups at 4 weeks of culture(P<0.05).To conclude,TiO2 nanotube layer can be loaded with silicon ions by plasma immersion ion implantation and deposition method and has good osteogenic activity.